Fibrosis and the associated decline in organ functionality lead to an almost 50% mortality rate in developed countries. Multipotent mesenchymal stromal cells (MSC) were shown to suppress the development and progression of fibrosis through secreted factors including specific non-coding RNAs transferred within extracellular vesicles (EV). However, age-associated chronic inflammation can provoke MSC senescence and change secretome composition, thereby affecting their antifibrotic properties. Alternatively activated macrophages (M2-type) are key players in chronic inflammation that may interact with MSC through paracrine mechanisms and decrease their antifibrotic functions. To confirm this hypothesis, we evaluated the M2-macrophage conditioned medium (CM-M2) effect on human adipose-tissue-derived MSC senescence in vitro. We found that CM-M2, as well as a pro-senescence agent, hydrogen peroxide (H₂O₂), increased p21+–MSC number and secretion of IL-6 and MCP-1, which are considered main senescence-associated secretory phenotype (SASP) components. Thus, both exposures led to the senescent phenotype acquisition of MSC. EV from both CM-M2 and H₂O₂-exposed MSC, which showed a decreased effect on the suppression of TGFβ-induced fibroblast-to-myofibroblast differentiation compared to EV from control MSC according to αSMA level and the αSMA+–stress fiber reduction. After two weeks of subsequent cultivation under standard conditions, MSC demonstrated a decrease in senescence hallmarks and fibroblast differentiation suppression via EV. These results suggest that M2-macrophage-induced chronic inflammation can reversibly induce MSC senescence, which reduces the MSC’s ability to inhibit fibroblast-to-myofibroblast differentiation.

Fibrotic focus is a pivotal morphofunctional unit in developing fibrosis in various tissues. For most fibrotic diseases, including progressive forms, the foci are considered unable to remodel and contribute to the worsening of prognosis. Unfortunately, the dynamics of the fibrotic focus formation and resolution remains understudied. A number of data suggest that the key cell type for focus formation are activated stromal cells marked by fibroblast activated protein alpha (FAPα) due to their high capacity for extracellular matrix (ECM) remodeling. We evaluated the dynamics of fibrotic focus formation and the contribution of the main cell types, including FAPα+ cells, in this process using a murine model of bleomycin-induced lung fibrosis. We revealed the very early appearance of FAPα+ cells in lungs after injury and assumed their important involvement to the myofibroblast pool formation. During the first month after bleomycin administration, FAPα+ cells colocalize with CD206+ M2 macrophages. Interestingly, during the reversion stage, we unexpectedly observed the specific structured foci formed by CD90+FAPα+ cells, which we suggested calling “remodeling foci”. Our findings highlight the crucial role of activated stromal cells in fibrosis initiation, progression, and reversion and provide emerging issues regarding the novel targets for antifibrotic therapy.

Intercellular communication is a critical component of maintaining tissue homeostasis. One of the communication methods is the transfer of a wide range of biologically active molecules - proteins, nucleic acids (primarily non-coding RNA) and lipids - in extracellular vesicles (EV). This allows EV-producing cells to change the metabolic and transcriptional activity of individual target cell populations over a wide range and “tune” it in accordance with tissue needs. Most of the existing studies are focused on the composition and functions of EVs secreted into the external environment, while the study of the membrane-associated vesicle subclass (membrane-associated vesicles, MAV) is hampered by the lack of generally accepted methods for their isolation from different cell types, including cultured ones, and characterization. In this paper, we present a protocol for isolating the MAV fraction of cultured mesenchymal stromal/stem cells while preserving the morphology and viability of the cells themselves using hyaluronidase treatment. This protocol demonstrated the highest efficiency compared to other tested methods and allows one to obtain a fraction significantly enriched in vesicles, as demonstrated by nanoparticle tracking analysis and transmission electron microscopy. A comparative analysis of the characteristics of MAV and EV secreted into the medium showed that the proposed MAV isolation protocol allows one to obtain a fraction with a similar particle concentration, but they are smaller in size compared to EV. Thus, the isolation method we described allows one to obtain a MAV fraction for further analysis of their composition and functional features.